We have been investigating Insertion Sequence (IS) movements in multidrug resistant bacteria with a focus on carbapenemase-producing Enterobacteriaceae (CPE). One Insertion Sequence, IS26, is frequently associated with resistance determinants, but its role remained unclear. We have previously analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the NIH Clinical Center, using target site duplications (TSDs) and their distribution patterns as guides, and discovered that a large fraction of plasmid reorganizations result from IS26 intramolecular replicative transpositions, including replicon fusions, DNA inversions, and deletions (1,2). We are interested in learning about the mechanisms behind these DNA rearrangements by the transposase encoded by IS26, and are currently studying its properties using recombinantly expressed protein in combination with various biochemical and biophysical assays. Another result that emerged from our analysis of CPE isolates from the NIH Clinical Center was the recognition of the central role played by Insertion Sequences that transpose using the so-call copy-out-paste-in mechanism (1,2). Despite its central importance, there is no current mechanistic information available regarding how this process works. Recently, we have identified a soluble and functional transposase from the IS256 family that is mobilized by this pathway, and we have been able to obtain diffracting crystals of the transposase complexed with various DNA substrates. We are currently analyzing the resulting structures of the transposase bound to transposon-end DNA to understand how it orchestrates this pathway of replicative DNA transposition. 1. He, Hickman, Varani, Siguier, Chandler, Dekker, and Dyda (2015) Insertion Sequence IS26 reorganizes plasmids in clinically isolated multidrug-resistant bacteria by replicative transposition. mBio 3, e00762-15. 2. He, Chandler, Varani, Hickman, Dekker, and Dyda (2016) Mechanism of evolution in high-consequence drug resistance plasmids. mBio 6, e01987.